Application of a primer combination for amplifying InDel molecular markers related to nitrogen uptake traits in maize in maize genotyping

Abstract

The present invention relates to the technical fields of molecular markers and molecular breeding, and in particular to the application of a primer combination for amplifying InDel molecular markers related to nitrogen uptake traits in maize in maize genotyping. The InDel molecular marker combination includes 7 InDel molecular marker sites of 5 nitrogen uptake-related candidate genes, and the primer sequences for amplifying the 7 InDel molecular marker sites are respectively as shown in SEQ ID NO: 43-44, SEQ ID NO: 53-54, SEQ ID NO: 35-36, SEQ ID NO: 61-62, SEQ ID NO: 9-10, SEQ ID NO: 1-2, SEQ ID NO: 25-26. By genotyping the maize GWAS natural population and screening through the genotyping results and classifying and comparing nitrate contents, the present invention can make the nitrate data results measured by candidate genes more reliable and can greatly improve the efficiency of screening maize germplasm resources with high nitrogen uptake efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers and molecular breeding, and specifically relates to the application of a primer combination for amplifying InDel molecular markers related to maize nitrogen absorption traits in maize genotyping. Background Art

[0002] my country is the second largest producer of corn in the world and the largest consumer of corn. It is well known that nitrogen fertilizer is an important fertility factor for increasing the yield of grain crops, and the appropriate increase in nitrogen fertilizer is of great significance to increasing yield. However, only about 21.6% of the large amount of nitrogen fertilizer input can be absorbed by corn and converted into agricultural productivity, and the rest is emitted into the atmosphere in the form of N2O or leached into the ground in the form of nitrates, causing a series of environmental problems. Therefore, how to improve the nitrogen use efficiency (NUE) of crops to ensure food and environmental security under low nitrogen fertilizer input is an important challenge in the future sustainable agricultural development.

[0003] In order to effectively improve the nitrogen utilization efficiency of crops and accurately regulate the growth and development of crops, it is necessary to conduct detailed research on the absorption, distribution and utilization mechanisms of nitrogen. However, the current reports on crop NUE traits are mostly concentrated in rice. The rice nitrogen efficient utilization gene OsNPF6.5 cloned by Chu Chengcai's team can achieve a 30% to 33% increase in yield compared to the control under the condition of halving nitrogen fertilizer, and a 30% increase in nitrogen fertilizer utilization efficiency. Qian Qian's team cloned an OsNR2 that encodes an NAD(P) (Htriphosphopyridine nucleotide)-dependent nitrate reductase. In addition, Xu Guohua's team has also cloned several rice NO3 genes, including OsNRT2.2, OsNRT2.3a, OsNRT2.4, OsNRT2.3b and OsLHT1. – Absorption and transport genes. It is precisely because more and more genes for efficient nitrogen utilization have been reported in rice that this provides great possibilities for saving nitrogen and increasing rice yield. However, there are relatively few studies on the mechanism of NUE in corn, which makes the research progress of corn nitrogen efficient breeding relatively slow, and there are few functional markers of nitrogen efficient absorption genes available.

[0004] With the continuous development of genome sequencing technology, genomic data resources have become increasingly abundant, making it much easier to develop InDel molecular markers through bioinformatics analysis. By designing specific primers based on the sequences flanking the insertion / deletion sites, the InDel molecular markers that reflect sequence length polymorphism through PCR (Polymerase Chain Reaction) amplification are stable in variation and highly accurate during the development process, which can avoid problems such as subsequent analysis ambiguity caused by specificity and complexity. InDel molecular markers can be used for genetic map construction, genetic diversity analysis, population structure evaluation, and core germplasm screening, etc. As high-throughput molecular markers, InDel markers have been widely applied in major crops such as rice, sorghum, and cotton. There are also many articles reporting on InDel molecular markers in maize, such as Zhang Tifu, Ge Min, etc. using InDel markers for the purity identification and analysis of maize hybrids.

[0005] However, there are relatively few reports on the development of InDel molecular markers related to nitrogen-efficient absorption in maize at present, which limits the application of InDel markers in maize nitrogen absorption-related research. Therefore, the development of InDel molecular markers related to maize nitrogen absorption traits can greatly accelerate the process of maize nitrogen-efficient breeding. Summary of the Invention

[0006] Aiming at the deficiencies of existing maize molecular markers in the development of nitrogen absorption functional markers, the purpose of the present invention is to provide the application of a primer combination for amplifying InDel molecular markers related to maize nitrogen absorption traits in maize genotyping. Using these molecular markers can greatly improve the efficiency of screening maize germplasm resources with high nitrogen-efficient absorption.

[0007] To achieve the above purpose, the present invention provides the following technical solutions:

[0008] The application of a primer combination for amplifying InDel molecular markers related to maize nitrogen absorption traits in maize genotyping, wherein the InDel molecular marker combination includes 7 InDel molecular marker sites of 5 nitrogen absorption-related candidate genes, and the primer sequences for amplifying the 7 InDel molecular marker sites are shown as SEQ ID NO: 43-44, SEQ ID NO: 53-54, SEQ ID NO: 35-36, SEQ ID NO: 61-62, SEQ ID NO: 9-10, SEQ ID NO: 1-2, SEQ ID NO: 25-26 respectively.

[0009] Among them, the 5 nitrogen absorption-related candidate genes are shown in the following table,

[0010]

[0011]

[0012] Among them, after the molecular marker performs PCR amplification on the maize genomic DNA, one allele can be amplified at the homozygous locus, and two alleles can be amplified at the heterozygous locus.

[0013] The present invention also provides a method for developing InDel molecular markers related to maize nitrogen uptake traits, comprising the following steps:

[0014] (1) Obtain maize genomic sequence information;

[0015] (2) Use the mInDel software to preprocess the maize genomic sequence, perform InDel analysis, primer design and evaluation;

[0016] (3) Screen better specific InDel molecular markers and perform PCR amplification on maize genomic DNA for verification.

[0017] Among them, the application of the primer combination for amplifying InDel molecular markers related to maize nitrogen uptake traits in maize genotyping comprises the following steps:

[0018] (1) Use the primers of the 7 InDel molecular markers to perform PCR amplification on maize genomic DNA;

[0019] (2) Perform gel electrophoresis detection on the amplification products;

[0020] (3) Read the amplified bands detected by gel electrophoresis to determine the genotype;

[0021] (4) Evaluate the nitrogen uptake ability of maize germplasm based on the genotype results.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention develops InDel (insertion-deletion) molecular markers related to maize nitrogen uptake traits. The forward and reverse primers corresponding to 7 loci of 5 nitrogen uptake-related candidate genes can accurately genotype the maize GWAS (Genome-wide association study) natural population. Through the screening of the genotyping results and the classification and comparison of nitrate contents, the nitrate data results measured by the candidate genes can be made more reliable, and the efficiency of screening maize germplasm resources with high nitrogen uptake efficiency can be greatly improved.

[0024] The InDel molecular marker related to nitrogen uptake traits in maize is a stable genetic variation, which is abundant in the maize genome and can be developed on a large scale. The PCR amplification products of the InDel molecular marker related to nitrogen uptake traits in maize can be detected by agarose gel electrophoresis and are convenient to use. Brief Description of the Drawings

[0025] Figure 1 It is a genotyping result diagram of a partial maize GWAS population using primer JS020.

[0026] Figures 2 - 8 It is a clustering diagram of nitrate content in the maize GWAS population. Detailed Embodiment

[0027] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0028] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods, and the experimental reagents and materials involved are all conventional biochemical reagents and materials unless otherwise specified.

[0029] (1) Obtaining the maize genome sequence

[0030] Download the genome sequence, protein sequence and gene annotation file of the V5 version of the maize whole genome from the EnsemblPlant (http: / / plants.ensembl.org / index.html) database.

[0031] (2) Prediction and verification of InDel molecular markers related to nitrogen uptake traits in maize

[0032] Import the maize genome data (in FASTQ format) into the mInDel software. After quality control of the data, perform InDel analysis. Design primers for InDel fragments with only two lengths in the B73 and HP maize materials with a nitrogen uptake difference of more than 3 times. After parental polymorphism testing, a total of 69 InDel molecular markers related to nitrogen uptake traits in maize were obtained, of which 32 could be detected for differences by 4% agarose gel electrophoresis, as shown in Table 1, and the primer sequences are shown in SEQ ID NO: 1-64.

[0033] Table 1 Primer sequences of 32 InDel molecular markers related to nitrogen uptake traits in maize and their predicted lengths of PCR products

[0034]

[0035]

[0036]

[0037] (3) Genotype analysis of maize

[0038] Genotype analysis was performed on 609 maize GWAS natural populations collected using 32 InDel molecular markers related to maize nitrogen uptake traits. The seedlings of the experimental materials were extracted using the Karroten DNA extraction kit for PCR amplification.

[0039] The PCR amplification system (15 μL) was as follows: 1 μL of template DNA (about 60 ng / μL), 1 μL of primer (10 mmol / μL), 7.5 μL of 2x Taq PCR Master Mix, and 4.5 μL of sterilized double-distilled water. Amplification was carried out on an Eppendorf PCR instrument, and the reaction conditions were: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, for a total of 35 cycles; and then extension at 72 °C for 2 min. The reaction products were electrophoresed on a 4% agarose gel, and the gel imaging was photographed.

[0040] (4) Application of evaluating nitrogen uptake ability in maize germplasm

[0041] PCR amplification was performed on 609 DNA materials in the GWAS population using 32 pairs of specific InDel molecular markers, and genotype analysis was carried out on the results. Those consistent with the genotype of the low-nitrogen uptake parental material B73 were denoted as A, and those consistent with the genotype of the high-nitrogen uptake parental material HP were denoted as B for classification and screening.

[0042] According to the genotype screening results, they were divided into B73 and HP categories, and the DNA material numbers of each category were counted and matched with the single-plant nitrate content data of the GWAS materials. It was found that among the 32 pairs of primers, 7 pairs of primers had different nitrate contents measured by classification and screening according to the genotype results. The 7 pairs of primers are shown in Table 2. Among them, the genotyping results of primer JS020 for part of the maize GWAS population are as Figure 1 shown. The clustering diagram of nitrate content in the maize GWAS population is as Figures 2 - 8 shown.

[0043] Table 2

[0044]

[0045] By screening the genotyping results and classifying and comparing the nitrate contents, the nitrate data results measured by candidate genes can be made more reliable, and the efficiency of screening maize germplasm resources with high nitrogen absorption can be greatly improved. This provides reliable genetic evidence for the use of functional markers in maize breeding for high nitrogen utilization and provides a molecular basis for cultivating maize varieties with high nitrogen efficiency.

[0046] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Use of a primer combination for amplifying InDel molecular markers related to nitrogen uptake traits in maize in evaluating the nitrogen uptake ability of maize germplasm, characterized in that: The InDel molecular marker combination described above includes 7 InDel molecular marker sites of 5 nitrogen uptake-related candidate genes, and the primer sequences for amplifying the 7 InDel molecular marker sites are shown as SEQ ID NO: 43-44, SEQ ID NO: 53-54, SEQ ID NO: 35-36, SEQ ID NO: 61-62, SEQ ID NO: 9-10, SEQ ID NO: 1-2, and SEQ ID NO: 25-26 respectively; The 5 nitrogen uptake-related candidate genes are shown in the following table:

2. Use of the primer combination for amplifying InDel molecular markers related to nitrogen uptake traits in maize according to claim 1 in evaluating the nitrogen uptake ability of maize germplasm, characterized in that, It includes the following steps: (1) Perform PCR amplification on maize genomic DNA using the primers of the 7 InDel molecular markers; (2) Detect the amplified products by gel electrophoresis; (3) Read the amplified bands detected by gel electrophoresis to determine the genotype; (4) Evaluate the nitrogen uptake ability of maize germplasm based on the genotype results.

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